逆转录病毒载体介导胸苷磷酸化酶在胰腺癌细胞表达

人胸腺嘧啶核苷磷酸化酶（ＴＰ）在一些肿瘤组织中活性增高,但其功能目前了解尚少。构建了表达ＴＰ的重组逆转录病毒载体,直接导入人胰腺癌ＰＣ－２细胞,ｍＰＣＲ扩增、Ｓｏｕｔｈｅｒｎ及Ｎｏｒｔｈｅｒｎ印迹和原位杂交证实转染细胞有外源ＴＰ的整合及表达,酶活性检测发现含外源ＴＰ细胞ＴＰ活性比ＰＣ－２细胞的内源性ＴＰ活性高ＴＰ活性高４０－７０倍,生长曲线和＾３Ｈ－ＴｄＲ参入率检测未发现含外源ＴＰ细胞生物学行为的     

外源基因在转基因动物中遗传和表达的稳定性

转基因技术经过近半个世纪的发展,已成为当今生物技术研究的热点。近10多年来,与核移植技术的结合,转基因效率大大提高,携带有不同外源基因的不同种类的转基因动物迅速增加。但是,成功获得转基因动物并不是转基因动物研究的最终目的,如何利用转基因技术为人类的需求服务才是科研人员始终面对的课题。在畜牧生产领域,通过转基因技术培育家畜新品种是转基因技术应用的重要体现,在我国这方面已经引起了广泛关注。但迄今为止,外源基因在转基因动物中遗传和表达的稳定性仍然是亟待解决的问题,究其原因,这主要与位置效应、外源基因的表观遗传学修饰和遗传效率相关,文章结合目前的研究进展和本实验室的研究结果,从这3方面阐述其作用机制,期望为转基因动物遗传育种向产业化的迈进提供一定的理论探讨。     

Production of biologically active human interleukin-4 in transgenic tobacco and potato

Interleukin-4 (IL-4) is a pleiotropic cytokine that plays a key regulatory role in the immune system. Recombinant human IL-4 (rhIL-4) offers great potential for the treatment of cancer, viral and autoimmune diseases. Unfortunately, the high production cost of IL-4 associated with conventional expression systems has, until now, limited broader clinical testing, particularly with regard to the more convenient and safer oral delivery of IL-4 as opposed to parenteral injection in patients. In this study, we investigated the feasibility of transgenic plants for the cost-effective production of rhIL-4. IL-4 expression vectors with different modifications under the control of a constitutive cauliflower mosaic virus 35S (CaMV 35S) promoter were introduced into tobacco by Agrobacterium-mediated transformation. Transgenic tobaccos expressing various levels of rhIL-4 protein were generated. Higher expression was achieved through IL-4 retention in the endoplasmic reticulum (ER), with the maximal accumulation being approximately 0.1% of total soluble protein (TSP) in the leaves. No improvement in expression was further achieved by replacing the native signal peptide of IL-4 with the plant signal peptide. The best rhIL-4-expressing vector shown in tobacco was selected and further transferred into potato plants. The analysis of transgenic tubers also revealed various levels of rhIL-4, with the highest being 0.08% of TSP. Sensitive in vitro T-cell proliferation assays showed that plant-derived rhIL-4 retained full biological activity. These results suggest that plants can be used to produce biologically active rhIL-4 and probably many other mammalian proteins of medical significance. Moreover, the production of plants expressing rhIL-4 will enable the testing of plant rhIL-4 by oral delivery for the treatment of clinical diseases.     

Enhancement of maize transformation efficiency by the use of maize matrix attachment regions

Improving genetic transformation efficiency is a major concern in plant genetic engineering. While various strategies have been investigated, the enhancement of selectable marker gene expression has not been tried extensively. We used maize matrix attachment regions (MARs) to bracket an herbicide resistance transgene, bar. MARs have been reported to enhance transgene expression level and stability. We show here that MARs not only enhance transformation efficiency by 50%, but are also able to increase or decrease relative efficiencies of each step of the regeneration process depending on MAR sequence combinations. Furthermore, we assessed the trans-effect of MARs in co-bombardment experiments with two independent plasmids, one including the MAR sequences and the other one the bar gene. As for simple bombardment, MARs enhanced transformation efficiency by having a positive influence on organogenesis step in the regeneration process.     

Targeted disruption of p53 attenuates doxorubicin-induced cardiac toxicity in mice

Use of the chemotherapeutic agent doxorubicin (Dox) is limited by dose-dependent cardiotoxic effects. The molecular mechanism underlying these toxicities are incompletely understood, but previous results have demonstrated that Dox induces p53 expression. Because p53 is an important regulator of the cell birth and death we hypothesized that targeted disruption of the p53 gene would attenuate Dox-induced cardiotoxicity. To test this, female 6–8 wk old C57BL wild-type (WT) or p53 knockout (p53 KO) mice were randomized to either saline or Dox 20 mg/kg via intraperitoneal injection. Animals were serially imaged with high-frequency (14 MHz) two-dimensional echocardiography. Measurements of left ventricle (LV) systolic function as assessed by fractional shortening (FS) demonstrated a decline in WT mice as early as 4 days after Dox injection and by 2 wk demonstrated a reduction of 31± 16% (P < 0.05) from the baseline. In contrast, in p53 KO mice, LV FS was unchanged over the 2 wk period following Dox injection. Apoptosis of cardiac myocytes as measured by the TUNEL and ligase reactions were significantly increased at 24 h after Dox treatment in WT mice but not in p53 KO mice. After Dox injection, levels of myocardial glutathione and Cu/Zn superoxide dismutase were preserved in p53 KO mice, but not in WT animals. These observations suggest that p53 mediated signals are likely to play a significant role in Dox-induced cardiac toxicity and that they may modulate Dox-induced oxidative stress.These two authors equally contributed to this study.     

Historical and contemporary gene dispersal in wild carrot (Daucus carota ssp. carota) populations

Background and Aims

Wild carrot is the ancestor of cultivated carrot and is the most important gene pool for carrot breeding. Transgenic carrot may be released into the environment in the future. The aim of the present study was to determine how far a gene can disperse in wild carrot populations, facilitating risk assessment and management of transgene introgression from cultivated to wild carrots and helping to design sampling strategies for germplasm collections.

Methods

Wild carrots were sampled from Meijendel and Alkmaar in The Netherlands and genotyped with 12 microsatellite markers. Spatial autocorrelation analyses were used to detect spatial genetic structures (SGSs). Historical gene dispersal estimates were based on an isolation by distance model. Mating system and contemporary pollen dispersal were estimated using 437 offspring of 20 mothers with different spatial distances and a correlated paternity analysis in the Meijendel population.

Key Results

Significant SGSs are found in both populations and they are not significantly different from each other. Combined SGS analysis indicated significant positive genetic correlations up to 27 m. Historical gene dispersal σ_g and neighbourhood size N_b were estimated to be 4–12 m [95 % confidence interval (CI): 3–25] and 42–73 plants (95 % CI: 28–322) in Meijendel and 10–31 m (95 % CI: 7–∞) and 57–198 plants (95 % CI: 28–∞) in Alkmaar with longer gene dispersal in lower density populations. Contemporary pollen dispersal follows a fat-tailed exponential-power distribution, implying pollen of wild carrots could be dispersed by insects over long distance. The estimated outcrossing rate was 96 %.

Conclusions

SGSs in wild carrots may be the result of high outcrossing, restricted seed dispersal and long-distance pollen dispersal. High outcrossing and long-distance pollen dispersal suggest high frequency of transgene flow might occur from cultivated to wild carrots and that they could easily spread within and between populations.     

Generation of a tightly regulated doxycycline-inducible model for studying mouse intestinal biology

To develop a sensitive and inducible system to study intestinal biology, we generated a transgenic mouse model expressing the reverse tetracycline transactivator rtTA2-M2 under control of the 12.4 kb murine Villin promoter. The newly generated Villin-rtTA2-M2 mice were then bred with the previously developed tetO-HIST1H2BJ/GFP model to assess inducibility and tissue-specificity. Expression of the histone H2B-GFP fusion protein was observed exclusively upon doxycycline induction and was uniformly distributed throughout the intestinal epithelium. The Villin-rtTA2-M2 was also found to drive transgene expression in the developing mouse intestine. Furthermore, we could detect transgene expression in the proximal tubules of the kidney and in a population of alleged gastric progenitor cells. By administering different concentrations of doxycycline, we show that the Villin-rtTA2-M2 system drives transgene expression in a dosage-dependent fashion. Thus, we have generated a novel doxycycline-inducible mouse model, providing a valuable tool to study the effect of different gene dosages on intestinal physiology and pathology.     

黄瓜转新型抗菌蛋白基因GNK2-1及其抗枯萎病的研究

GNK2-1为一种来自银杏（Ginkgo biloba）种仁的新型抗真菌蛋白,具有较强的真菌抗性且性质稳定。序列分析表明,其结构与所有已知的抗真菌蛋白不同,而与富含半胱氨酸的植物类受体激酶的胞外结构域相似。为探索GNK2-1基因在黄瓜（Cucumis sativus）抗病反应中的作用,利用基因重组技术构建了GNK2-1的高效组成型表达载体,并利用根癌农杆菌（Agrobacterium tumefaciens）介导转入黄瓜栽培品种农城3号（Cucumis sativus＇ Nongcheng No.3＇）基因组中。通过对获得的抗性植株进行PCR、RT-PCR和Western blot检测分析,结果表明GNK2-1基因可在T0代转基因植株中转录表达,并能在T1代转基因黄瓜中稳定遗传。离体枯萎病抗性鉴定结果表明,转GNK2-1基因的黄瓜对枯萎病的抗性增强,GNK2-1可以作为黄瓜抗病性改良的潜在基因资源。